Immunolocalization of thymosin alpha 1, thymopoietin and thymulin in mouse thymic epithelial cells at different stages of culture: a light and electron microscopic study.

The secretory evolution of the thymic hormones (thymulin, thymosin alpha 1 and thymopoietin) in cultured thymic reticuloepithelial cells (TREC) was studied by immunocytochemical techniques using monoclonal anti-thymulin or anti-thymosin alpha 1 and polyclonal anti-thymopoietin antibodies (Ab). The culture of TREC was performed with a medium where L-valine was replaced by D-valine, thus ensuring rapid and selective development of these cells. The number of thymulin, thymosin alpha 1 or thymopoietin-containing cells increased progressively from Day 6 to Day 12 of the culture. The localization of the three thymic hormones within the TREC also varied according to the age of the culture. By light microscopy the staining of the three hormones was localized in some cytoplasmic granules at the beginning of the culture and at Day 90, while at Day 12 it was throughout the cytoplasm. In electron microscopy these localizations corresponded respectively to vacuoles of different sizes and to cytosol. All these results show that the synthesis and excretion of thymulin, thymosin alpha 1 and thymopoietin evolve during the development of TREC in culture.     

Thymic hormone-containing cells. VI. Immunohistologic evidence for the simultaneous presence of thymulin,thymopoietin and thymosin α1 in normal and pathological human thymuses

The localization of the three best-defined thymic hormones, namely, thymulin, thymopoietin and thymosin α1 was studied by immunofluorescence using antibodies directed against these three molecules. With both human thymus frozen sections and cultured cells, thymic hormones were found exclusively in the epithelial component (recognized by its keratin content), in normal as well as pathological thymuses. The double-labeling experiments using the different anti-thymic hormone antibodies showed that the same epithelial cells contained the three hormones. These results suggest that the production of different hormones in the thymus is accomplished by the same epithelial cells.     

Do epidermal cells produce thymic hormones in vivo? An immunohistochemical study using anti-thymic hormone antibodies

Cultured human epidermal cells have been shown to produce soluble factors endowed with T cell differentiating activities. In addition, the presence of thymopoietin and FTS/thymulin-like factors has been reported in normal human and mouse epidermis using immunohistochemical methods and anti-thymic hormone antibodies. The present study was conducted to re-evaluate the presence of thymic hormones in normal epidermal cells using a panel of monoclonal antibodies (mAbs) and rabbit antisera to several well characterized thymic factors. The reactivity of the following antibodies was tested by indirect immunofluorescence on human and mouse tissue sections: a) two anti-FTS/thymulin mAbs; b) one anti-FTS/thymulin rabbit antiserum; c) one anti-thymopoietin rabbit antiserum; d) one anti-thymosin alpha 1 mAb. Our results show that: 1) all five antibodies reacted with human and/or mouse thymic epithelial cells; 2) none of the 3 antithymic hormone mAbs (2 anti-FTS/thymulin and 1 anti-thymosin alpha 1 mAbs) reacted with normal skin; 3) only 2 out of 5 antibodies, namely the anti-FTS and anti-thymopoietin antisera cross-reacted with mouse and human epidermis and labeled keratinocytes, as previously reported; these latter 2 antibodies also stained nude mouse epidermal cells and labeled non-thymic, non-epidermal normal mouse epithelial tissues, suggesting that the cross-reactive epitope is common to a number of epithelial cells; 4) the antigen defined by the anti-FTS and anti-thymopoietin antisera was not related to keratins, since absorption experiments using purified human epidermal keratins failed to abolish staining of the epidermis. We conclude from this study that epidermal cells do not produce in vivo the well characterized thymic hormones: FTS/thymulin, thymopoietin and thymosin alpha 1. The precise nature of the antigenic structure recognized within epidermis by the anti-FTS and anti-thymopoietin antibodies remains to be defined.     

An enzyme immunoassay for synthetic thymulin

Thymulin, a metallononapeptide with the following aminoacid sequence: pyroGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-AsnOH is a thymic hormone involved in T cell differentiation requiring zinc to express biological activity as measured by the rosette assay. We established an enzyme immunoassay (EIA) for synthetic zinc-free thymulin with a thymulin-acetylcholinesterase conjugate as tracer and specific polyclonal rabbit antithymulin antibodies. The assay is performed as a classical competition assay in microtiter plates previously coated with mouse monoclonal IgG to rabbit IgG. A quantitative thymulin assay more sensitive than radioimmunoassays (RIAs) previously described was obtained with a sensitivity (IC50) of 32.5 +/- 5 pg/ml and a detection limit of 5 pg/ml. Analysis in the EIA of synthetic thymulin analogs showed that the minimal peptidic structure necessary for enzymatic tracer competition is the C-terminal part Lys3 to Asn9. It was also shown that the biologically active form of thymulin (zinc-bound) has the same immunoreactivity as zinc-free thymulin and that other thymic hormones, thymosin alpha 1 and thymopoietin II (or TP5) and unrelated short peptides do not cross-react with thymulin. These data demonstrate the specificity of this EIA for thymulin and show its suitability for application in biological fluids.     

Production of a lymphocyte differentiating factor (ELDIF) by cultured human epidermal cells

T lymphocyte maturation activity of supernatants from cultured human epidermal cells was investigated using a biological rosette assay based upon Thy 1 induction on T cell precursors. Between days 5 and 18 of keratinocyte cultures, supernatants exhibited Thy 1-inductive properties. Optimal activity was found between days 10 and 14 of culture, which correspond to the beginning of epidermal cell stratification. This activity was independent of the presence of prostaglandin E. Absorption experiments using monoclonal and polyclonal antibodies showed that ELDIF was different from other recognized thymic hormones (thymulin, thymopoietin, thymosin alpha 1), produced by thymic epithelial cells and known to induce T cell markers.     

Lack of reactivity of anti-human immunodeficiency virus (HIV) P17/18 antibodies against alpha 1 thymosin and of anti-alpha 1 thymosin monoclonal antibody against P17/18 protein

The blood rate of alpha 1 thymosin is increased during HIV infection, despite the thymus involution. Anti-alpha 1 thymosin antibodies inhibit HIV replication in vitro. A homology between alpha 1 thymosin and the HIV P17/18 core protein exists and would explain a cross-antigenicity. We have studied the interaction between anti P17/18 antibodies from HIV patients and alpha 1 thymosin and between an anti-alpha 1 thymosin monoclonal antibody and the P17/18 protein. We were unable to confirm any cross-reactivity. During acquired immune deficiency syndrome, a major involution of the thymus appears with a severe depletion of thymocytes and epithelial cells. Certain thymic functions are missing, as corroborated by the reduction of the hormone thymulin in the blood. At the same time, the blood rate of the 2 other hormones (partly of thymic origin), alpha 1 thymosin and beta 4 thymosin is increased. One of the theories explaining this discordance is that patients with acquired immunodeficiency syndrome produce molecules which have a cross antigenicity with these thymic hormones. Sarin et al. have recorded a 50% homology between the C-terminal part (last 18 aminoacids) of alpha 1 thymosin and the part between the 92nd and the 109th aminoacids of the HIV P17/18 protein. The cross reactivity between this P17/18 protein and alpha 1 thymosin would explain the high rates of alpha 1 thymosin found in the radio-immunoassay of sera from patients infected with HIV. Another result of this cross-reactivity is the ability of alpha 1 thymosin antibodies to inhibit HIV replication in the H9 permissive cell line.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of molecules detected by three different anti-H-2Kk monoclonal antibodies.

Three different monoclonal antibodies specific for murine Class I H-2Kk-encoded determinants have been found to bind to molecules other than the classical 45 Kd Class I glycoprotein. These results were obtained after electrophoresis of a detergent lysate of spleen cells, and extraction of molecules present in multiple gel fractions. The antigenic activity in each gel fraction was assessed after removal of detergent by capacity to inhibit the binding of each of these antibodies to Ig-capped spleen cells in a rosetting assay. Only the H100-27R9 antibody was inhibited by an extract representing 45-50 Kd proteins resembling Class I molecules. This antibody also bound material extracted from a higher 65-70 Kd fraction of the gel. The binding of the 11-4.1 and H100-30R3 antibodies was inhibited by unique molecules in the molecular weight range of less than 20 Kd. The inhibitory material was not sensitive to pronase but was sensitive to glycosidases. This material could be absorbed by each of the H100-30R3 and 11-4.1 antibodies but not by H100-27R9. All data are consistent with unique, low molecular weight carriers of carbohydrate-defined epitopes which can bind monoclonal antibodies having specificity for H-2K-encoded gene products. It is predicted that these are glycolipids, but it is not yet known whether they are cell-surface or cytoplasmic molecules.     

Myasthenic sera recognize the human acetylcholine receptor bound to thymopoietin

The thymic hormone, thymopoietin (Tpo), from human (HTpo), bovine (BTpo) and from synthetic (sHTpo) origins bound to the acetylcholine receptor (AChR) solubilized by Triton 1.5% from human muscle. This binding was demonstrated either by inhibition of formation of radiolabeled alpha bungarotoxin (alpha Bgt)-AChR complexes measured after precipitation by ammonium sulfate or by a myasthenic serum containing a high concentration of anti-AChR antibodies, or directly by incubating the human AChR with radiolabeled sHTpo or BTpo. The 125I-labeled alpha Bgt-AChR complexes were totally inhibited by 10(-6) M sHTpo or BTpo. The complexes formed by AChR and the radiolabeled Tpo were recognized specifically by sera containing anti-AChR antibodies from myasthenic patients. The active pentapeptide derivative of Tpo, thymopentin, another thymic hormone, thymulin, as well as bovine insulin did not interfere with the specific binding of alpha Bgt to human AChR. Tpo and anti-AChR antibodies could participate together in the inhibition of neuromuscular conduction with Tpo modulating the depressive effect of the antibodies on the neuromuscular junction in myasthenia gravis.     

Evidence for the direct action of thymulin on avian NK cells

The ability of thymulin to directly enhance NK cell-mediated cytotoxicity was examined. Specific cell population depletions were done in K and SLD chicken splenocyte preparations using anti-CD3, CD4, and CD8 monoclonal antibodies and secondary complement-fixing polyclonal antibodies. The remaining cells were incubated overnight with in vitro treatments of thymulin and IFN-gamma, either separately or together, followed by an assay for cytotoxicity. Although the control K-strain had higher overall NK cell-mediated cytotoxicity than the thymulin-deficient SLD-strain, the following trends were seen in both strains. Thymulin continued to enhance NK activity following CD4 or CD3 cell depletion, but not after CD8 or CD8 and CD4 cell depletion. Since avian NK cells express CD8 alpha, but not CD3 or CD4 on their surface, these results suggest that the ability of in vitro thymulin treatments to enhance NK activity is not mediated by T-cells but may be due to direct effects on NK cells.     

Murine lupus monoclonal antibodies define five epitopes on two different Sm polypeptides.

Monoclonal anti-Sm (Smith) antibodies derived from the mouse strain MRL/lpr were isolated and characterized by binding to purified antigen, immunoprecipitating characteristic uridine-rich RNAs from Hela cell extracts, and by Western blot analysis using rabbit thymus extract. Five different Sm epitopes were demonstrated by epitope blockade and probing Western blots with the monoclonal antibodies. Human anti-Sm serum inhibited each monoclonal antibody from binding to antigen, indicating that both human and mouse antibodies bind to the same Sm epitopes. Human anti-Sm antibodies bound to 28,000 and 16,000 MW polypeptides, a small number also binding to a 14,000 MW polypeptide. The monoclonal antibodies also bound to the 28,000 and/or the 16,000 polypeptide, and provided evidence to suggest that these two Sm polypeptides bear some structural similarities, but are distinct molecules.     

Monoclonal antibodies to sheep MHC class II molecules recognize all HLA-D or subsets of HLA-D region products

Six murine monoclonal antibodies raised against sheep MHC class II molecules were analyzed for reactivity with HLA-D subregion products. All the antibodies reacted with human peripheral blood lymphocytes, monocytes, and B-lymphoblastoid cell lines homozygous for various HLA-DR specificities, suggesting that the antibodies recognized nonpolymorphic determinants on HLA class II molecules. SDS-PAGE and two-dimensional NEPHGE/SDS-PAGE analyses of molecules immunoprecipitated from 35S-methionine-labeled, DR-homozygous B-lymphoblastoid cell lines showed that the monoclonal antibodies precipitated typical class II molecules (Mr 32-34K and 25-29K). From comparison with antibodies of known HLA-D subregion specificity, two of the sheep antibodies appeared to react with the products of single HLA-D subregions, while another showed balanced reactivity with all HLA-D molecules. Antibody SBU.II 38-27 reacted exclusively with HLA-DQ molecules, antibody SBU.II 28-1 with HLA-DP molecules, and antibody SBU.II 49-1 with HLA-DR, -DQ, and -DP molecules. However, analysis of immunoprecipitates from surface-iodinated WT-49 cells (DR3 homozygous) using SBU.II 28-1 and the DP-specific monoclonal antibody B7/21, suggested that the two antibodies immunoprecipitated different alpha polypeptides. The two antibodies SBU.II 38-27 and 28-1 appear to be at least as specific as existing reagents, if not more so. As such, they are of value in their potential contribution to our understanding of the molecular characteristics and ultimately the functions of the HLA-DQ and -DP subregion products, as well as the identification/characterization of HLA-D equivalents in other species.     

Natural mouse IgG reacts with self antigens including molecules involved in the immune response.

IgG isolated on protein A-Sepharose from pools of normal sera from various mouse strains were examined by immunoblotting for reaction with self antigens. Homogenates of the major mouse organs, i.e. brain, skin, spleen, kidney, adrenals, thymus, heart, muscle and liver were used as the source of autoantigens. IgG stained at least 220 bands on the immunoblots. The antigens corresponding to these bands were tentatively identified by molecular mass estimation and referenced to computerized mouse protein data banks. IgG mainly recognized enzymes but it also stained intracellular structural constituents and surface molecules implicated in the functioning of the immune system. The validity of this identification was confirmed by analyzing purified antigens from mouse or other animal species by immunoblotting and enzyme immunoassays. Furthermore, extracts of 125I-surface-labeled cells were immunoprecipitated with IgG in the liquid phase or immobilized on beads. The proteins precipitated migrated to the same positions as those precipitated by specific monoclonal antibodies (mAb), such as class I alpha chain and beta 2-microglobulin, class II alpha and beta chains, CD3, CD4 and CD8 antigens. The results obtained with several enzyme immunoassay procedures using cell membrane extracts, specific mAb and normal IgG further supported the specific interaction of IgG with Ia, CD4 and CD8 molecules. Affinity chromatography indicated that at least 20% of normal mouse IgG possess polyreactive autoantibody function. Dissociation constants of these IgG were calculated for some autoantigens and found to be in the range of 2 x 10(-6)-7 x 10(-6) M. It is concluded that normal mouse IgG exhibit autoreactivities similar to those previously described for IgM.     

Feedback regulation of the secretion of a thymic hormone (thymulin) by human thymic epithelial cells in culture

The production of the thymic hormone, thymulin (FTS), was studied in primary cultures of human thymic epithelium by immunofluorescence using monoclonal anti-thymulin antibodies. The number of thymulin-containing cells and the thymulin level in the culture supernatant increased gradually during the culture. Addition of synthetic thymulin to the culture medium reduced significantly the increase of thymulin-containing cells. Conversely, addition of monoclonal anti-thymulin antibody from the beginning of the culture exacerbated the spontaneous increase of thymulin-containing cells and abrogated the effects of thymulin. Combined with similar data previously reported in vivo, these results demonstrate that thymulin is actively produced by cultured thymic epithelial cells and that its synthesis can be down-regulated by the hormone itself.     

Thymopoietin, a polypeptide ligand for the alpha-bungarotoxin binding site in brain: an autoradiographic study.

Thymopoietin, a 48-49-amino acid polypeptide present in the thymus gland, was investigated as a potential ligand for the neuronal nicotinic alpha-bungarotoxin binding site in rat brain. Binding of [125I]alpha-bungarotoxin to whole rat brain sections was inhibited by thymopoietin in a concentration-dependent manner with an IC50 of 30.0 +/- 8.2 nM as compared to 1.1 +/- 0.3 nM for alpha-bungarotoxin. However, at concentrations of thymopoietin of up to 1 microM, [3H]nicotine binding to high affinity sites was not inhibited. Thysplenin, a polypeptide with considerable homology to thymopoietin did not affect [125I]alpha-bungarotoxin binding. These results suggest that thymopoietin selectively interacts with the nicotinic alpha-bungarotoxin binding site labelled by [125I]alpha-bungarotoxin rather than the neuronal nicotinic receptor(s) labelled by [3H]nicotine. Autoradiographic studies revealed that 1 microM thymopoietin almost completely inhibited [125I]alpha-bungarotoxin binding in all brain regions. Computer-assisted image analysis of displacement curves was performed on various brain areas rich in alpha-bungarotoxin binding, such as the dorsal endopiriform nucleus, fields 1 and 2 of Ammon's horn, the polymorph cell layer of the dentate gyrus and cortical layers 4 and 5. Thymopoietin inhibited [125I]alpha-bungarotoxin binding with similar potency in all these regions, suggesting that it interacted at the same site in the different brain areas. The IC50 values averaged over the six regions were 24.6 +/- 2.8 nM for thymopoietin and 1.2 +/- 0.2 nM for alpha-bungarotoxin. These results show that thymopoietin specifically interacted with the alpha-bungarotoxin site with a similar potency in different brain regions. It is suggested that thymopoietin represents a selective ligand for alpha-bungarotoxin binding sites in brain.     

Thymic endocrinology.

Thymus endocrinology is characterized by the action of various hormones on the thymus endocrine milieu consisting of thymocytes, thymic epithelial cells and thymic stromal cells. Extrathymic hormonal influences include pituitary-derived hormones, such as prolactin and indirectly by ACTH via hydrocortisone from the adrenal, by thyroid-stimulating hormone (TSH) via thyroid hormones from the thyroid, and by LH and RH via sex steroids from gonads and adrenal. In addition, the thymus produces several putative thymic hormones: thymosin alpha 1, thymulin and thymopoietin, which have been reported to circulate and to act on both prothymocytes and mature T-cells in the periphery thus maintaining their commitment to the T-cell system and its functions. These endocrine influences decline with age and are associated with "thymic menopause" and cellular immune senescence contributing to the development of diseases in the aged. The intrathymic environment is characterized by a complex network of paracrine and autocrine endocrine signals involving both interleukins and thymic peptides. Thymic epithelial cells respond to IL-1 with proliferation and secretion of IL6 and GM-CSF. They similarly respond to cellular interactions with the production of IL1. Thymic epithelial cells also secrete thymic hormones, as exemplified by the zinc-thymulin complex, under stimulation with IL1 and other hormonal influences. Thymic stromal cells contribute, at minimum, IL1. These various interleukin and thymic hormone influences can be envisioned to operate in a synergistic interactive network to carry the evolving T-cell through its stepwise development to a mature T-cell.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptors for human interferon alpha on bovine cells: specificity and tissue distribution

Human interferon alpha 1 (HuIFN alpha 1) is known to protect bovine as well as human cells against viral infection. Hence, we investigated the specificity and tissue distribution of receptors for HuIFN alpha 1 on various cells. [35S]HuIFN alpha 1 bound specifically to homogenates of bovine tissues and particularly to bovine liver, but there was also specific binding to spleen, kidney, brain, adrenal gland, lung, thymus, skeletal muscle, heart, mammary gland and testis. There was no difference in the degree of binding of HuIFN alpha to foetal or adult liver. Competitive binding experiments showed that bovine interferon alpha C (BoIFN alpha C) competed with HuIFN alpha 1 for binding to a bovine liver plasma membrane preparation, indicating that these two IFNs bind to the same receptor. An 35S-labelled IFN alpha 1-receptor complex was isolated from bovine liver extracts by SDS/polyacrylamide gel electrophoresis, and shown to have a molecular weight of 153 kDa. Isolation of the bovine IFN alpha receptor would be a feasible approach to the characterization of the HuIFN alpha receptor.     

Retinal pigment epithelial cell plasma membrane: a monoclonal antibody study

The plasma membranes of retinal pigment epithelial cells are highly specialized organelles with multiple functions including nutritional and metabolic support of the photoreceptor cells. Using purified bovine retinal epithelial cell plasma membranes as antigen, we produced two monoclonal antibodies, MAbD1-C6 and MAbD1-C8, that cross react with the plasma membranes from bovine, rat and human retinal pigment epithelial cells. In radioimmunoassay both MAbD1-C6 and MAbD1-C8 had similar affinities for bovine plasma membranes. Both monoclonal antibodies identified a protein of 72 Kd with an apparent subunit of 32-35 Kd. The protein was localized to the cell surface of human and bovine retinal pigment epithelium by immunocytohistochemistry. In the normal eye the antigen identified by the monoclonal antibodies was strongly associated with the retinal pigment epithelium and weakly associated with lens tissue. Using either monoclonal antibody, components of purified bovine or rat retinal pigment epithelial plasma membranes were precipitated from solution. Based on these results, we conclude that both monoclonal antibodies are closely related and that they may be useful for the isolation and study of retinal pigment epithelial cell structure.     

Generation and characterization of the murine monoclonal antibody M-KID 2 to VLA-3 integrin.

Through the analysis of the antigenic phenotype of a recently established human renal carcinoma cell line (KJ29), we have demonstrated that alpha 3 subunit of the integrin family is selectively expressed by the plastic adherent cell subpopulation. Because of the scanty availability of monoclonal antibodies to this adhesion molecule, we have used KJ29 cell line as immunogen to raise novel murine monoclonal antibodies. We isolated an hybridoma secreting the mAb M-KID 2 of the IGg1k isotype that immunoprecipitates from intrinsically [35S]-Methionine labeled KJ29 cells, an heterodimer of 130/130 and 110/150 Kd, in reducing and nonreducing conditions respectively. This reactivity was completely abolished by immunodepletion of the cell extract with a polyclonal anti alpha 3 chain antiserum. Treatment of M-KID 2 immunoprecipitates with various solutions of pH ranging from 2 to 10.5, to dissociate alpha 3 from beta 1 chains, showed a retention of both alpha 3 beta 1 chains thus indicating that the epitope identified by mAb M-Kid 2 is likely to be constituted by the alpha 3 beta 1 heterodimer. Furthermore immunohistochemical studies on selected frozen and paraffin embedded tissues with mAb M-Kid 2 have provided staining pattern indicating the recognition of Vla-3. These findings demonstrate that mAb M-KID 2 can represent a valuable reagent for the study of Vla-3 integrin in normal and pathologic conditions.     

The in vitro effect of the thymic factor thymopoietin on a subpopulation of lymphocytes from severely malnourished children.

Several investigators have shown that the proportion of T cell lymphocytes was greatly reduced while that of the B cell lymphocytes was virtually unaffected; thus the relative proportion of null cells was significantly increased in severely malnourished patients. Other studies have demonstrated that extracts of bovine thymus will induce an increase in the proportion of T cell rosettes from patients with various disorders. This study has demonstrated that peripheral blood lymphocytes from severely malnourished patients with secondary infections contain a subpopulation of lymphocytes which will respond in vitro in the presence of thymopoietin by increased numbers of E rosettes. These results also support the concept of a finite population of responder cells and indicate that other thymopoietin insensitive subpopulations of null cells are increased when the proportion of T cells are greatly suppressed. Further characterization of the lymphocyte subpopulation(s) affected by thymopoietin is needed.